Recombinant human factor IX and use thereof

ABSTRACT

The present invention aims at converting factor IX into a molecule with enhanced activity which provides an alternative for replacement therapy and gene therapy for hemophilia B. Using recombinant techniques, factor IX having substitution of amino acid residue of SEQ ID NO: 7 at amino acid position selected from the group consisting of 86, 277, and 338 (exclude the circumstance of a single substitution at amino acid position 338) exhibits better clotting activity than recombinant wild type factor IX.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to a Provisional Application(Application No. 60/884,129) filed on Jan. 9, 2007, which is herebyincorporated by reference in its entirety.

Although incorporated by reference in its entirety, no arguments ordisclaimers made in the parent application apply to this Non-provisionalapplication. Any disclaimer that may have occurred during theprosecution of the above-referenced application(s) is hereby expresslyrescinded. Consequently, the Patent Office is asked to review the newset of claims in view of all the prior art of record and any search thatthe Office deems appropriate.

FIELD OF THE INVENTION

The present invention relates to a recombinant human factor IX protein,and a nucleic acid sequence encoding thereof. The present invention alsorelates to a method for treating hemophilia.

BACKGROUND OF THE INVENTION

In the developed world, the current therapeutic choices for treatment ofhemophilia patients comprise prophylactic and on-demand replacementtherapy (Lofqvist T. et al., J. Intern. Med. 1997; 241:395-400) witheither plasma-derived or recombinant coagulation factor concentrate(Lippert B. et al., Blood Coagul. Fibrinolysis. 2005; 16:477-485).Standard treatment for Hemophilia is infusions of protein concentratesto replace the defective clotting factor. The amount infused dependsupon the severity of bleeding, the site of the bleeding, and the bodysize of the patient. People with severe forms of the disease may betreated by regular prophylactic infusions. The outcome is good withtreatment and management so most people with hemophilia are able to leadrelatively normal lives. However, the high cost and limited availabilityof the recombinant protein make dosing of clotting factors a crucialissue in the treatment of hemophilia. In addition, the plasma-derivedproducts run the risk for HIV and hepatitis B and C transmission, andthe half-life of the infused protein in a patient is short, whichresults in the necessity for fairly frequent infusions (White G. C. etal., Transfus. Sci. 1998; 19:177-189).

Other treatments, such as gene therapy and tissue implant techniques arealso under study as possible treatments (Hough C. et al., J. Thromb.Haemost., 2005; 3:1195-1205). One clinical trial of gene therapy inhemophilia patients showed only transient therapeutic increments ofclotting factor expression due to generation of antibody againstdelivering vehicle (Manno C. S. et al., Nat. Med. 2006; 12:342-347).Therefore, gene therapy remains an investigational method with manyobstacles to overcome before it can be widely used as treatment forhemophilia.

Hemophilia B is caused by a deficiency of a blood plasma protein calledfactor IX that affects the clotting property of blood. The disorder iscaused by an inherited X-linked recessive trait, with the defective genelocated on the X chromosome. Thus, the disorder occurs primarily inmales. Hemophilia B occurs in about 1 out of 30,000 men.

Human factor IX is a vitamin K-dependent zymogen which plays animportant role in blood coagulation. Factor IX circulates as a 415-aminoacid single chain zymogen with a molecular mass of 55,000 daltons and ispresent in normal plasma at approximately 5 μg/ml.

Recombinant factor IX products offer greatly reduced risk for HIV andhepatitis B and C transmission. If recombinant factor IX with enhancedclotting activity can be generated through genetic engineering of factorIX DNA, it will not only lower the cost for the clotting factor but alsoreduce the dose of it in managing patients with hemophilia. Moreover,this method will also provide a more efficient tool for gene therapytrials in patients with hemophilia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the SDS-PAGE analysis of purified recombinant factor IX.Purified proteins (4 μg/lane) from Wild type (IX-7) and mutants IX-1,IX-2, IX-8, IX-3, IX-4, IX-5 and IX-6 were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were rununder non-reducing conditions. Factor IX protein bands were visualizedby Commassie blue staining. The concentration of affinity-purifiedrecombinant wild type and alanine-replaced factor IX was determined bythe BCA™ protein assay kit (Pierce, Rockford, Ill., USA) using bovineγ-globulin (cat.#23212, Pierce) to generate a standard curve forcalculating the protein concentrations.

FIG. 2 shows human factor IX levels in hemophilia B mice at 24 hoursafter hydrodynamic treatment with pBS-HCRHP1-A-FIX (see “Method”). Malehemophilia B mice (20 μg body weight) were subjected to hydrodynamicshock by tail vein injection of 2 ml of 50 μg DNA in 6-8 seconds. Themice were recovered and sacrificed 24 h after injection for collectionof blood plasma for clotting activity and protein level determination(IX:Ag) by Enzyme-linked ImmunoSorbent assay (ELISA). Each barrepresents the data of individual mouse. Standard curves for ELISA andfor clotting activity were derived in parallel experiments from serialdilutions of normal plasma pooled from 20 healthy donors. The factor IXprotein level and clotting activity in the pooled plasma were assumed tobe 5 μg/ml.

FIG. 3 shows results of factor IX expression by adeno-associated virus(AAV)-mediated gene transfer to hemophilia B mice. Construction of therAAV2/8 vector carrying, individually, IX-6 and IX-7 was described in“Methods”. Homozygous female hemophilia B mice (n=6, aged 8˜14 weeks,weighing 16˜18 g) were respectively injected intravenously with 4×10¹²vg/kg (a) 4×10¹¹ and 8×10¹⁰ vg/kg (b) of the rAAV2/8 vehicles carryingIX-6 and IX-7 as indicated. Blood was collected 2 weeks after injectionfor measurement of factor IX protein level and clotting activity. Eachgroup of mice was numbered according to age. Specific clotting activitypresented as percentage was defined as the clotting activity measuredfor each sample divided by its mass (IX:Ag) measured by ELISA. Standardcurves for ELISA and for clotting activity were derived in parallelexperiments from serial dilutions of normal plasma pooled from 20healthy donors. Factor IX protein level and clotting activity in thenormal pooled plasma was assumed to be 5 μg/ml.

SUMMARY OF THE INVENTION

The present invention provides a recombinant human factor IX proteinhaving substitution of amino acid residue of SEQ ID No: 7 at amino acidposition selected from the group consisting of 86, 277 and 338, providedthat a single substitution at amino acid position 338 is excluded.

The present invention further provides an isolated nucleic acid encodinga recombinant human factor IX protein having nucleic acid sequence shownin SEQ ID NO: 9, 10, 11, 12, 13 or 14.

The present invention further provides a pharmaceutical compositioncomprising: (a) a human factor IX protein of the present invention, and(b) a pharmaceutically acceptable carrier, excipient, or diluent.

The present invention further provides a method for generating a humanfactor IX protein of the present invention in vivo comprising: (a)constructing a vector carrying a nucleic acid encoding the human factorIX protein; and (b) administering the vector to a mammalian.

The present invention further provides a method for treating hemophiliacomprising administering to a patient in need of such treatment with aneffective amount of the protein of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a recombinant factor IX with higheractivity than wild type factor IX, and it allows less protein injectionthan the latter into hemophilia to reach the therapeutic level.

The present invention provides a new factor IX DNA sequence that wouldexpress a factor IX protein with a higher clotting activity than the DNAsequence expressing wild type factor IX when delivered into animals byvarious means such as viral vectors.

Using recombinant techniques, factor IX with simultaneously double ortriple alanine replacement at positions 86, 277, and 338 exhibited 2˜14times better clotting activity than wild type recombinant factor IX(IX-7). In an attempt to understand the causes contributing to theincreased clotting activity of these factor IX variants, severalfunctional parameters were determined. Table 1 demonstrates that theincreased clotting activity was factor VIIIa-dependent and wasattributed to the enhanced affinity of factor IX for factor VIII and anincreased k_(cat) and decreased K_(M) for factor X.

Accordingly, the present invention provides a recombinant human factorIX protein having substitution of amino acid residue of SEQ ID No: 7 atamino acid position selected from the group consisting of 86, 277 and338, provided that a single substitution at amino acid position 338 isexcluded. The present proteins have alanine residue substitution atamino acid sequence of wild type factor IX, wherein the substitution isat the amino acid position selected from the group consisting of 86,277, and 338, provided that a single substitution at amino acid position338 is excluded.

The term “amino acid” used herein is a molecule that contains both amineand carboxyl functional groups. In biochemistry, this term refers toalpha-amino acids with the general formula H₂NCHRCOOH, where R is anorganic substituent. In the alpha amino acids, the amino and carboxylgroups are attached to the same carbon, which is called the α-carbon.The various alpha amino acids differ in which side chain (R group) isattached to their alpha carbon. In general, the standard amino acidssuch as alanine, asparagine, aspartic acid, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, methionine, phenylalanine,serine, threonine, tryptophan, tyrosine or valine can be used as asubstituted amino acid. In a preferred embodiment, the substituted aminoacid residue is alanine.

In a more preferred embodiment, the protein of the present invention hasSEQ ID No: 1, 2, 3, 4, 5 or 6. In the most preferred embodiment, theprotein of the present invention has SEQ ID No: 6.

The substitution creates the non-naturally occurring human factor IXexhibiting from 2 to 14 folds more than wild type factor IX protein'sclotting activity (Table 1). The protein has enhanced affinity forcofactor, factor VIIIa. In an embodiment, the recombinant human factorIX protein has K_(d) values of 0.1˜0.5 nM. In an embodiment, therecombinant human factor IX protein has K_(M) values of 32˜128 nM,preferably 32˜56 nM (Table 2).

The present invention also provides an isolated nucleic acid encoding arecombinant human factor IX protein having nucleic acid sequence shownin SEQ ID NO: 9, 10, 11, 12, 13 or 14. In a preferred embodiment, thenucleic acid has SEQ ID NO: 11, 12 or 13. In a more preferredembodiment, the nucleic acid has SEQ ID NO: 14.

The present invention further provides a pharmaceutical compositioncomprising: (a) a human factor IX protein of the present invention; and(b) a pharmaceutically acceptable carrier, excipient, or diluent.

Besides in vitro expression and analysis of their biological properties,the present invention also relates to using mouse model to evaluate thepotential role of the alanine variants. The hemophilia B mouse offers amodel that allows the physiological role of these variant factor IXs tobe studied. To estimate the degradation pathway of factor IX, thesehuman factor IX proteins is injected into the tail vein of hemophiliamouse and the characteristics of these mutant proteins is observed.

Hydrodynamics-based delivery of naked plasmid DNA to liver can generatetherapeutic plasma levels of transgene products in mice. This technologyis used to deliver the DNA encoding the wild type and variant factor IXgenes to hemophilia B mice.

Systemic delivery of therapeutic transgenes by viral vectors canpotentially lead to long-term transgene expression (Miao C. H. et al.,Mol. Ther. 2001; 3:947-957). But the efficiency of gene transfer tohepatocytes is poor. The recombinant AAV delivery system garneredenthusiastic support when it demonstrated efficacy in initialpreclinical studies in the hemophilia B animal models (Davidoff A. M. etal., Mol. Ther. 2005; 11:875-888). Two clinical studies were initiatedusing an AAV serotype 2 vector to deliver factor IX to the muscle (MannoC. S. et al., Blood. 2003; 101:2963-2972) by direct intro muscularinjection and to the liver via hepatic artery infusion (Manno C. S. etal., Nat. Med. 2006; 12:342-347). In the muscle trial, plasma level offactor IX generally did not raise above 1%, whereas levels of up to 12%were detected in the plasma of single patient treated by hepatic arterydelivery of the AAV vector. However the increase was followed by atransient rise in serum transaminase levels and loss of factor IXexpression, probably due to pre-existing host immunity to AAV capsidproteins that targeted the transduced cells. Because the recombinantIX-6 has higher activity than wild type (IX-7) in our mouse model, itcould be possible to reduce the injection quantity of viral particlesand reach the same factor IX activity.

Accordingly, the present invention also provides a method for generatinga human factor IX protein of the present invention in vivo comprising:

-   (a) constructing a vector carrying a nucleic acid encoding the human    factor IX protein; and-   (b) administering the vector to a mammalian.

In a preferred embodiment, the mammalian is human.

The term “administration” used herein is not limited but includes viavector, plasmid, liposome, DNA injection, electroporation, gene gun,intravenously injection or hepatic artery infusion.

The present invention further provides a method for treating hemophiliacomprising administering to a patient in need of such treatment with aneffective amount of a protein of the present invention.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The embryos, animals, andprocesses and methods for producing them are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Modifications therein and other uses will occurto those skilled in the art. These modifications are encompassed withinthe spirit of the invention and are defined by the scope of the claims.

EXAMPLE

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

Materials

Purified human factors VIa, XIa, Xa, and X, and polyclonal goatanti-human factor IX antibodies (cat. #GAFIX-AP) were purchased fromEnzyme Research Laboratory (ERL, South Bend, Ind., USA).Phosphatidylcholine (PC), phosphatidylserine (PS) and ITS(Insulin-Transferrin-Sodium selenite) media supplements used in serumfree media were from Sigma Chemical Co. (St. Louis, Mo., USA).Preparation of tissue factor and PCPS phospholipids were describedpreviously (Chang Y J, et al., Biochemistry, 1999:10940-10948). FactorIX deficient plasma, Spectrozyme FXa, and Spectrozyme FIXa werepurchased from American Diagnostica Inc. (Greenwich, Conn., USA). Allthe restriction endonucleases and polymerases were products of the NewEngland Biolabs, Inc. (Beverly, Mass., USA). Geneticin (G418) was fromCalBiochem (Merck KGaA, Darmstadt, Germany). A Vmax microtiter platereader equipped with a thermal controller (Molecular Devices Corp.,Menlo Park, Calif., USA) was used for all spectrophotometric assays.QAE-Sephadex A50, and Resource Q were from Amersham (Amersham PharmaciaBioTech, UK, Bucks, England).

Methods

In-Vitro Mutagenesis, Expression and Purification of Factor IX

Site-specific mutagenesis was performed on the human factor IX CDNA by aPCR-based method (QuickChange, Stratagene, Boston, USA) using primerpairs for replacing the codons at residues 86, 277 or 338 with those foralanine. The sequences of the primers are IX86(5′GTGAATTAGATGCAACATGTA3′) pairing with IX86T7(5′GTTACATGTTGCATCTAATTCAC3′) for replacing residue 86; IX277(5′GAACTGGACGCACCCTTAGTGC3′) pairing with IX277-2(5′CTAAGGGTGCGTCCAGTTCCAG3′) for replacing residue 277, and IX338(5′CATGTCTTGCATCTACAAAG3′) pairing with IX338D(5′CTTTGTAGATGCAAGACATG3′) for residue 338. With these primer pairs, 7different factor IX cDNA's were generated, fully sequenced, and togetherwith wild type factor IX (IX-7) subcloned into pCR3™-Uni (Invitrogen,Calif., USA) for expression in human 293 cells under the control ofcytomegalovirus. Of the 7 recombinant factor IX mutants, three aresingle replacement mutants with alanine substitution at residues 86, 277or 338 (SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO: 8, respectively),another three are double replacement mutants with alanine substitutionsat 86 plus 277 (SEQ ID NO: 3), 86 plus 338 (SEQ ID NO: 4) and 277 plus338 (SEQ ID NO: 5), and the other is triple replacement mutant with 86,277 and 338 all replaced by alanine (SEQ ID NO: 6). Identification ofcell clones expressing the recombinant proteins was by ELISA (see below)using the commercially available factor IX specific antibodies.Expansion of the correct cell clones in serum free media to largequantity and purification of the recombinant proteins were performed asdescribed previously. Briefly, one liter of the cultured supernatant wasadjusted to 5 mM benzamidine and 4 mM EDTA and subjected tocentrifugation at 4,350 rpm (HA6000, Sorvall RC-3C Plus, DuPont) for 30mm at 4° C. After removal of cell debris, the supernatant was passedthrough a QAE-Sephadex A50 column (30 ml, 5×2.5 cm) equilibrated withIBS/EDTA (20 mM Tris-Cl, pH 7.5, and 150 mM NaCl/4 mM EDTA). Afterwashing the column with 1 liter of TBS containing 5 mM benzamidine,factor IX was eluted with a gradient of 0˜30 mM CaCl₂ in TBS/1 mMbenzamidine. Fractions containing factor IX were identified by ELISA(see below), pooled, dialyzed against 100-fold excess volumes of 50 mMTris-Cl, pH 7.5, and filtered through a 0.22-μm syringe filter(Millipore, Cork, Ireland). Approximately 100 ml of the QAE-eluent wasloaded on to a Resource Q column (1 ml, Akta FPLC purifier, AmershamBiosciences). After washing in buffer containing 50 mM Tris-Cl, pH 7.5,the column was eluted with a gradient of NaCl (0˜1 M) in 50 mM Tris-Cl,pH 7.5. Factor IX was eluted at fractions of approximate 250 mM NaCl.The purified recombinant factor IX proteins were verified by SDS-PAGEfollowed by staining with Coomassie blue.

Enzyme-Linked Immunosorbent Assay (ELISA) and Clotting Activity Assay.

The protein mass of wild type and the alanine-replaced human factor IX(hFIX) in cultured supernatant and in mouse plasma was determined byELISA using polyclonal antibodies to human factor IX. The antibodies arespecies-specific polyclonal anti-hFIX antibodies that do not cross-reactwith murine factor IX. Pooled plasmas of at least 20 healthy individualswere used in serial dilutions in parallel experiments to preparestandard curves for calculation of the level of factor IX. The standardcurves from the pooled plasma paralleled to those derived from thepurified plasma factor IX (IMMUNINE, Baxter AG, Vienna, Austria).

The function of the recombinant factor IX proteins was determined bytheir clotting activities in the one-stage activated partialthromboplastin time (aPTT) assays. Pooled normal plasma in serialdilutions was used as assay standards. The standard curves of theclotting assays parallel to those derived from the purified plasmafactor IX (IMMUNINE) and the recombinant factor IX (BeneFIX, GeneticsInstitute, Inc. Cambridge Mass., USA). The specific activity of a samplewas defined as the clotting activity divided by its mass. The factor IXprotein mass and clotting activity in pooled normal human plasma wasassumed to be 5 μg/ml.

Activation of Factor IX by Factor XIa and by Factor VIIa and TissueFactor (VIIa·TF) Complex

The activation of factor IX by factor XIa in the presence of TBS/5 mMCaCl₂ was performed at an enzyme to substrate ratio of 1:200. Aliquotsfrom the reaction mixtures were withdrawn at timed-intervals, placed ina solution containing 1% SDS and 2 mM EDTA to stop the reaction,adjusted in gel loading buffer (final concentrations: 50 mM Tris-Cl, pH6.8, 2 mM EDTA, 1% SDS, 8% glycerol, and 0.025% Bromophenol blue), andsubsequently subjected to SDS-PAGE analysis. The activation of factor IXby the VIIa·TF complex was performed at an enzyme to substrate ratio of1:80 and in TBS/5 mM of CaCl₂. Aliquots from the reaction mixtures werewithdrawn at timed-intervals and resolved by SDS-PAGE analysis.

Interaction of Factor IXa with Factor X

Factor IXa was prepared by activation with factor XIa as describedabove. The concentration of the factor IXa molecules was determined bytitration with antithrombin III (ATIII) as described previously (Chang YJ, et al., Journal of Biological Chemistry. 2002; 277:25393).Interaction of factor IXa with factor X was analyzed by monitoringkinetically the hydrolysis of Spectrozyme FXa by activated factor Xgenerated by factor IXa in the absence and presence of factor VIIIa.Briefly, when without factor VIIIa, factor IXa (10 nM) was incubatedwith PCPS at room temperature for 5 min before different concentrationsof factor X (0-1 μM) and 0.5 mM Spectrozyme FXa were added and thereaction was recorded as the absorbance change of the mixture accordingto the incubation time. The final volume was 100 μl and the reaction wasperformed at 37° C. in buffers containing 0.01% BSA and 40 μM PCPS inTBS/5 mM CaCl₂ (TBS/CaCl₂/BSA/PCPS). The interaction of factor IXa withfactor X in the presence of factor VIIIa was performed by incubation of25 μl of factor VIIIa (freshly prepared) with an equal volume of FIXadiluted and preincubated in TBS/CaCl₂/BSA/PCPS, and a 50 μl of areaction mixture containing PCPS, Spectrozyme FXa and factor X addedsubsequently. The factor Xa activity generated by the factor IXa-factorVIIIa complex (the intrinsic tenase activity) in the mixture wasdetected kinetically on the microtiter plate reader. Finalconcentrations were 0.25 nM for wild type or mutant factor IXa, 0.4 nMfor factor VIIIa, 40 μM for PCPS, 0-200 nM for factor X, and 0.5 mM forSpectrozyme FXa. The factor IXa activity was calculated by the followingequation as described previously. Absorbance (A₄₀₅)=at²+bt+c

Interaction of Factor IXa with Factor VIIIa

Binding experiments were performed by monitoring the intrinsic tenaseactivity at limiting concentrations of factor VIIIa. Twenty-fivemicroliters of freshly prepared factor VIIIa (0.4 nM) were incubatedwith 25 μl of different concentrations of wild type or mutant factor IXa(0-20 nM) to form the intrinsic tenase complex. Activity of theintrinsic tenase complex formed by binding of factor VIIIa to factor IXawas then measured by the addition of 50 μl of a mixture of factor X andSpectrozyme FXa in a reaction buffer containing TBS/Ca/PCPS/BSA. Finalconcentrations were factor VIIIa, 0.1 nM, PCPS, 40 μM, factor X, 100 nM,Spectrozyme FXa, 0.5 mM, and factor IXa, 0˜15 nM. Experiments wereperformed in duplicate for 3 independent reactions, and curves werefitted using all data points. The K_(d) values were derived bycalculations according to the following equation as described previously(Chang Y J, et al., Journal of Biological Chemistry. 2002; 277:25393).

$\left\lbrack {{IXa} - {VIIIa}} \right\rbrack = {\frac{\lbrack{IXa}\rbrack_{t} + \lbrack{VIIIa}\rbrack_{t} + {Kd}}{2} - \frac{\sqrt{\begin{matrix}{\left( {\lbrack{IXa}\rbrack_{t} + \lbrack{VIIIa}\rbrack_{t} + {Kd}} \right)^{2} -} \\{{4\lbrack{IXa}\rbrack}_{t}\lbrack{VIIIa}\rbrack}_{t}\end{matrix}}}{2}}$Interaction of Factor IX with Antithrombin III (ATIII)

The inhibition of factor IXa by ATIII was performed by gel analysis ofthe enzyme-inhibitor complex. 0.53 μM factor IXa and 0.53 μM ATIII wereincubated at 37° C. in 100 μl of TBS/5 mM CaCl₂ with or without 0.01unit/ml heparin. Aliquots (20 μl) of the reaction mixtures werewithdrawn at different time intervals (0, 5, 10, 20, and 30 min), mixedwith gel loading buffer (final concentration: 50 mM Tris-Cl, pH 6.8, 2mM EDTA, 1% SDS, 8% glycerol, and 0.025% Bromophenol blue) and developedby SDS-PAGE followed by staining with silver.

Animal Experiments

All the animal experiments described below followed standard procedures.Animals were treated according to the guidelines of the National TaiwanUniversity in Taiwan or the National Institutes of health guidelines foranimal care and the guidelines of the Children's Hospital and RegionalMedical Center at Seattle, USA. Hemophilia B mice of C57BL/6 strainbackground were originally obtained from Dr. Darrel Stafford (BiologyDepartment, University of North Carolina at Chapel Hill, N.C., USA) andDr. Katherine High (The Children's Hospital of Philadelphia, Pa., USA).

Protein Infusion into Hemophilia B Mice

Mice were anesthetized with 2.5% Avertin and injected intravenously with0.25 μg/g body weight of recombinant proteins IX-6 (SEQ ID NO. 6), IX-7(SEQ ID NO. 7) and IX-8 (SEQ ID NO. 8) to reach a hypotheticalcirculating level of 5 μg/ml, assuming the total plasma volume of amouse be estimated by 1/10 of body weight. At 5 min, 15 min and 2 hafter injections, mice were sacrificed and blood was collected into 3.8%sodium citrate (9:1 v/v) from the inferior vena cava. Plasma wereprepared by centrifugation at 8,000 rpm (Eppendorf, 5415R) at roomtemperature for 10 minutes and subjected to analyses for protein mass byELISA and clotting activity by aPTT assay.

Hydrodynamic Injection and Measurement of Human Factor IX Expression inHemophilia B Mice

The cDNA's coding for recombinant factor IX (SEQ ID Nos. 9-16) wereindividually subcloned into pBS-HCRHP1-A (Miao C. H. et al., Mol. Ther.2001; 3:947-957), for optimal expression in the liver of hemophilia Bmice under the control of the human apolipoprotein E/C-I gene locuscontrol region and the human alpha-1 antitrypsin promoter, as well asthe truncated human factor IX intron 1 and the bovine growth hormonepolyadenylation signal sequence for improved protein expression. Micewere anesthetized with 2.5% Avertin and aliquots (50-100 μg) of theseexpression plasmids (pBS-HCRHP1-A-FIX) were dissolved in 2 ml PBS(phosphate-buffered saline) and injected into the tail vein of 17˜24 gmice over a period of 6˜8 seconds. Each experiment with respectiveplasmid was repeated several times with at least two different batchesof plasmid DNA prepared at different times. Blood samples were takenfrom inferior vena cava when necessary and made into plasma by dilutionwith 1/10 (v/v) of 3.8% sodium citrate. Human factor IX protein levelsand clotting activity expressed and present in the mouse plasma weremeasured by ELISA and aPTT, respectively. Liver tissues were alsoprepared and measured for factor IX expression by ELISA. Total liver(1˜1.7 g) were homogenized in the presence of 2 ml of T-PER tissueprotein extraction reagent (cat. #78510, Pierce) containing 5 μl/ml ofprotease inhibitor cocktail (Sigma). After centrifugation, the totalprotein in the supernatant of the extract was about 31.8±3.5 mg/ml whichwas subjected to ELISA for human factor IX levels.

Gene Transfer Experiments Using Pseudotyped ssAAV2/8 Vector

The coding sequences of IX-6 (SEQ ID NO: 14), and IX-7 (SEQ ID NO: 15)cDNA (1.4-kb in length, without 3′ untranslated region) wereindividually subcloned into pBS-HCRHP1-A, subsequently excised as a4.3-kb SpeI fragment containing the entire expression cassette(enhancer, promoter, factor IX coding region and intron 1, and bovinegrowth hormone polyadenylation signal) and subcloned into pAAV-MCS(Stratagene, La Jolla, Calif.) at the NotI site converted to XbaI byligation with linkers. The resultant plasmids consist of the entireexpression cassette (4.3-kb SpeI fragment) flanked by the invertedterminal repeats (ITRs) of AAV2. The ssAAV2/8 vector carrying individualIX-6 and IX-7 expression cassettes were used to produce AAV viralparticles by the Triple transfection method as previously described(Xiao X, et al. J. Virol. 1998; 72:2224-2232). The vector titers weredetermined by quantitative polymerase chain reaction (LightCycler 480,Roche Applied Science, Mannheim, Germany) using factor IX specificprimers (forward: 5′-GGAAGCAGTATGTTGATGG-3′ and reversed:5′-TGGTTCACAGGACTTCTGGT-3′) and expressed as vector genome (vg)/ml. Tailvein administration of viral particles into hemophilia B mice wasperformed with vector doses of 4×10¹², 4×10¹¹ or 8×10¹⁰ vg/kg bodyweight. These mice were sacrificed 2 weeks after tail vein injection ofAAV particles. Blood samples were collected from inferior vena cava forplasma preparation. Human factor IX protein levels and clottingactivities expressed and present in the mouse plasma were measured byELISA and aPTT, respectively.

Results

Engineered Recombinant Factor IX Proteins Exhibited Higher ClottingActivity than Wild Type Factor IX (IX-7)

To search for factor IX variants with augmented clotting function, weselected 3 amino acid positions 86, 277, and 338, of human factor IX andreplaced them singly or in combination with alanine. We expressed atotal of 7 alanine-replaced factor IX variants in human 293 kidneycells. The expression levels of the mutated factor IX were quiteequivalent to that of the wild type factor IX (IX-7) and approximated0.08˜0.5 μg/24 h/10⁶ cells. After purification, all the recombinantfactor IX proteins revealed as a single band in SDS-PAGE with a mobilitysimilar to that of plasma derived factor IX (FIG. 1). The identity ofthe recombinant proteins were verified and confirmed by amino acidsequence analysis revealing the first 5 residues of each recombinant(data not shown). The integrity of the IX-7 and alanine mutants wasfurther investigated by activation of the recombinant factor IX proteinswith factor XIa and with the factor VIa and tissue factor (VIIa·TF)complex, and inhibition by antithrombin III in the presence and absenceof heparin. These experiments revealed no apparent differences betweenIX-7 and all the 7 alanine recombinants (data not shown). We concludethat alanine substitutions at positions 86, 277 and 338 did not alterthe global structure of factor IX.

The clotting activity of the purified factor IX recombinants was shownin Table 1. Recombinant IX-7 was fully active and had a specificactivity of 94%. All the factor IX mutants were also functional and had1.1˜13 times more clotting activity than IX-7. Among all, factor IX-6was the most active and had 13 times better than IX-7's clottingactivities.

TABLE 1 Specific Clotting activity of purified factor IX.^(a) Clottingact. Sp. Act. (μg/ml) (%) IX-7 4.72 ± 0.81  94 ± 16 IX-1 5.38 ± 0.50 115± 10 IX-2 5.96 ± 0.77 130 ± 15 IX-8 18.08 ± 0.14  362 ± 3  IX-3 6.34 ±0.34 122 ± 7  IX-4 8.51 ± 2.06 199 ± 41 IX-5 9.38 ± 3.30 188 ± 66 IX-666.04 ± 2.84  1293 ± 57  ^(a)The recombinant factor IX was purified from0.5~1 liters cultured supernatant of stably-transfected HEK 293 cells.The purified factor IX concentration was determined by Biuret method(BCA protein assay kit, Pierce) as described in legend to FIG. 1 anddiluted to 5 μg/ml. The clotting activity was determined by the aPTTassays. Pooled normal human plasma in serial dilutions was used togenerate the standard curves. The factor IX concentration in the pooledplasma was estimated to be 5 μg/ml. The specific activity of a samplewas defined as the clotting activity divided by its mass inconcentration and presented as percentage. The experiments were repeated2 times.The Increased Clotting Activity of IX-6 Correlated with its Affinity forFactor VIIIa.

Enzymatic kinetics parameters were measured with the factor IXarecombinants to investigate the influence of the alanine substitution onthe clotting function of factor IXa. We assume that factor tenaseactivity is proportional to the concentration of the factor IXa incomplex with factor VIIIa (FIXa·FVIIIa). Therefore, one could monitorthe generation of factor Xa by the FIXa·FVIIIa complex as an indicationfor the binding affinity of factor IXa for factor VIIIa and calculatethe apparent dissociation constant (K_(d)). As shown in Table 2, theK_(d)'s for the three variants with single alanine mutations (IX-1, IX-2and IX-8) and one of the variants with double mutations (IX-3) (1.20nM˜1.95 nM) are approaching that for IX-7 (2.44 nM). Surprisingly, theK_(d)'s for the two with double mutations (IX-4 and IX-5) and the onewith triple mutations (IX-6) (0.4 nM, 0.34 nM and 0.19 nM, respectively)were significantly lower than those for the IX-7 or the three singlemutations. There is a 10-fold dramatic difference in K_(d) between IX-6and IX-7. Moreover, it appears that IX-6 and factor VIIIa can form anefficient enzyme complex (0.144 nM) as compared with IX-7 and factorVIIIa (0.033 nM). These consequences, together with the better affinitybetween IX-6 and factor VIIIa than IX-7 and factor VIIIa, seem tojustify the increased clotting activity of IX-6.

In the absence of factor VIIIa, the kinetic parameters, i.e., K_(M) andk_(cat) for all the recombinant mutants and IX-7 were very similar(Table 2), with k_(cat)/K_(M) around 240.59˜726.81 M⁻¹sec⁻¹. The resultindicates that without factor VIIIa, factor IXa is a rather inefficientenzyme in cleaving factor X, which has been observed for activated IX-7and so does for these factor IXa mutants.

TABLE 2 Kinetic parameters of factor Xa generation in the absence andpresence of factor VIIIa. K_(M) V_(max) Enzyme^(a) K_(cat) ^(b) ×k_(cat)/K_(M) K_(d) Without FVIIIa nM nM FXa/min nM 10⁻⁴ (M⁻¹sec⁻¹) nMIX-7 756.8 ± 102.6 0.16 ± 0.04 10 2.62 ± 0.59 352.95 ± 109.17 IX-1 531.9± 130.2 0.14 ± 0.03 10 2.25 ± 0.50 426.54 ± 35.84  IX-2 395.3 ± 43.0 0.07 ± 0.05 10 1.22 ± 0.86 303.54 ± 204.5  IX-8 479.8 ± 44.0  0.08 ±0.04 10 1.37 ± 0.61 280.05 ± 108.00 IX-3 290.1 ± 54.58 0.12 ± 0.05 101.95 ± 0.77 726.81 ± 298.62 IX-4 479.0 ± 87.5  0.08 ± 0.02 10 1.35 ±0.41 278.40 ± 36.18  IX-5 681.4 ± 151.1 0.10 ± 0.07 10 1.64 ± 1.11240.59 ± 161.64 IX-6 742.8 ± 18.0  0.17 ± 0.05 10 2.82 ± 0.85 380.68 ±121.26 K_(M) V_(max) Enzyme^(a) K_(cat) ^(b) × k_(cat)/ K_(d) With 0.4nM FVIIIa^(c) nM nM FXa/min nM 1 K_(M) × 10⁸ nM IX-7 54.04 ± 5.25  27.81± 0.66  0.033 14.17 ± 0.34  2.64 ± 0.21 2.44 IX-1 55.39 ± 10.57 15.11 ±0.90  0.049 5.09 ± 0.30 0.93 ± 0.12 1.42 IX-2 44.27 ± 2.15  29.74 ±0.42  0.056 8.89 ± 0.13 2.01 ± 0.07 1.20 IX-8 44.79 ± 8.33  28.41 ±3.34  0.052 9.08 ± 1.07 2.04 ± 0.14 1.32 IX-3 35.17 ± 3.19  34.12 ±1.13  0.039 14.56 ± 0.48  4.16 ± 0.28 1.95 IX-4 80.95 ± 13.15 46.78 ±8.02  0.106 7.36 ± 1.26 0.91 ± 0.04 0.40 IX-5 116.53 ± 12.45  148.81 ±14.03  0.114 17.93 ± 6.72  2.12 ± 0.44 0.34 IX-6 46.01 ± 10.53 52.26 ±10.22 0.144 6.07 ± 1.19 1.33 ± 0.10 0.19 ^(a)The concentration of factorIXa-VIIIa complex was derived from experimental conditions and observedK_(d) ^(b)k_(cat) = V_(max)/[enzyme], the unites are M FXa, M⁻¹FIXa(orFIXa-FVIIIa), s⁻¹ ^(c)The reaction was incubated with 0.4 nM factorVIIIa and 0.25 nM factor IXa to activate 0-200 nM factor X in thepresence of 0.5 mM Spectrozyme FXa, 40 μM PCPS and 5 mM Ca2+. # ND: notdeterminedRecombinant IX-6 was More Active than IX-7 In Vivo.

To evaluate the effectiveness of the recombinant factor IX in vivo, weinfused IX-6, IX-7, and IX-8 proteins into hemophilia B mice andfollowed the protein levels and clotting function of the proteins byanalyzing the plasma samples at timed intervals. As shown in Table 3,when approximately 1 μg/ml of IX-7 protein was detected in the plasma ofthe hemophilia B mice at 5 min after tail vein injection of 10 μgrecombinant protein in 20 g mice, the level decreased to 76% initiallevels at 15 min and to 18% initial at 2 h. The circulating factorIX-7's clotting activity at each timepoint ranged around 121˜483%protein mass. Parallel experiments with IX-6 and IX-8 revealed thatwhile comparable to IX-7's protein levels were detected at each timepoint in mice infused with IX-8 and IX-6, the mice infused withrecombinant IX-8 exhibited 2.4 (5 min's sample) ˜3.67 (2 h's) times,respectively, more clotting function than those infused with IX-7. Inconsistent with in-vitro's experimental findings with IX-6, hemophilia Bmice infused with IX-6 exhibited a much higher than IX-7's activity,approximately 2.6˜7.5 times more than IX-7's specific activitythroughout each time point [IX-7 versus IX-6, (2.91/1.15) versus(12.05/0.72) for 5 min's data as an example].

TABLE 3 Time course study of plasma factor IX levels in mice infusedwith recombinant factor IX proteins.* IX-7 (n = 4) IX-8 (n = 2) IX-6Clotting act. IX:Ag Clotting act. IX:Ag Clotting act. IX:Ag μg/ml (% offirst time point)  5 min. 2.91 ± 1.2  1.15 ± 0.2 (100) 6.98 ± 1.4  0.92± 0.1 (100) 12.05 ± 4.4   0.72 ± 0.1 (100) (n = 5)  15 min. 2.63 ± 1.00.88 ± 0.3 (76) 5.24 ± 2.7 0.86 ± 0.1 (94) 6.74 ± 1.4 0.55 ± 0.2 (77) (n= 4) 120 min 0.64 ± 0.4 0.21 ± 0.2 (18) 2.35 ± 0.2 0.36 ± 0.0 (39) 4.80± 3.1 0.45 ± 0.1 (62) (n = 3) *Approximately 10 μg factor IX wasinjected into each mouse to reach hypothetical protein concentration of5 μg/ml plasma. Blood samples were taken at different time points afterinjection and subjected to ELISA and clotting assay. Standard curveswere derived from serial dilutions of normal plasma in parallelexperiment. ELISA system used pAb from ERL (Gafix-AP160) as coatingantibody and ERL pAb-HRP (Gafix-HRP) as detecting antibody. Normalplasma factor IX concentration is assumed to be 5 μg/ml.Gene Delivery to Hemophilia B Mice by Hydrodynamic Injection Method

The efficacy of the factor IX mutants for the treatment of hemophilia Bwas also evaluated by the method of hydrodynamic injection of expressionplasmids carrying individual cDNAs into hemophilia B mice for expressionof the factor IX mutants predominantly by the liver. The expressionlevels were measured 24 h after DNA injection and shown in FIG. 2 andTable 4. Recombinant IX-7 and all the alanine replacement mutants exceptIX-6 were expressed and secreted into plasma at a protein level of0.7˜1.17 μg/ml and clotting activity of 0.46˜2.26 μg/ml. IX-6 hadreproducibly higher protein levels (3.22±0.58 μg/ml, n=4) and clottingactivities (12.34±2.87 μg/ml, n=4) than IX-7 and the other alaninemutants. Interestingly, IX-6 also had 6 times (clotting activity) and 2times (specific activity, i.e., IX:Ag/clotting) higher activity thanIX-8. The higher factor IX protein levels found in mice injected withIX-6 DNA than with IX-7 and the other mutants was not observed with thecell culturing expression system which indicated that IX-7 and IX-6 weresynthesized to similar levels. To further investigate possibleexplanations for the higher plasma factor IX protein levels in the miceinjected with IX-6 than with IX-7 DNA, we extracted and quantified theintracellular and circulating factor IX in the liver and plasma,respectively. As shown in Table 4 approximately equal amount of factorIX was extracted from the liver of mice treated with IX-6 and IX-7 DNA(1.18 and 1.36 μg/g liver, respectively, p=0.43). These data indicatedthat IX-6 DNA- and IX-7 DNA-injected mice had comparable amount ofintracellular factor IX in the liver. Interestingly, the IX-6DNA-treated mice had statistically more circulating factor IX than IX-7DNA-treated mice (1.5 times difference, p<0.01, n=7˜10). Moreimportantly, the plasma clotting activity of IX-6 DNA-mice is 15 timeshigher than that of the IX-7 DNA-treated mice (4.88±2.12 μg/ml vs0.31±0.15 μg/ml, Table 4).

TABLE 4 Factor IX levels in mice hydrodynamically injected withplasmids.* plasma Liver Clotting act. (μg/ml) Total protein (mg) IX:Ag(μg) IX:Ag (μg/ml) (Sp. Act. %) IX-7 (n = 7) 60.92 ± 7.02 1.34 ± 0.760.20 ± 0.08 0.31 ± 0.15 (154 ± 33) IX-6 (n = 10) 65.73 ± 7.76 1.60 ±0.61 0.34 ± 0.10 4.88 ± 2.12 (1411 ± 357) *Hydrodynamic delivery offactor IX expression plasmids into hemophilia B mice. Male hemophilia Bmice of 20 μg were subjected to hydrodynamic shock by tail veininjection of 2 ml of 100 μg DNA in 6-8 s. The mice were recovered andsacrificed 24 h after injection for collection of blood plasma forclotting assay by aPTT and protein levels by ELISA (IX:Ag). For ELISAsystem, plates were coated with pAb from ERL (Gafix-AP160) and ERLpAb-HRP (Gafix-HRP) was used as detecting antibody. Standard curve wasderived from serial dilutions of normal plasma. Normal plasma factor IXconcentration is assumed to be 5 ug/ml.Gene Transfer Experiments Using Pseudotyped ssAAV2/8 Vector

The efficacy of IX-6 in gene therapy was further investigated usingviral vectors. The hemophilia B mice were injected intravenously withrecombinant adeno-associated viral vectors (rAAV2/8) carrying individualIX-6, and IX-7 at a dose of 4×10¹² vg/kg. At two weeks after injection,the plasma factor IX protein level expressed by rAAV2/8 carrying IX-7was about 2.3˜7.8 μg/ml (n=5) (FIG. 3 a). Compared with IX-7, the factorIX level expressed by rAAV2/8 carrying IX-6 reached 1.58˜4.23 μg/ml(n=6). It appears that rAAV2/8 carrying IX-6 had lower factor IX proteinlevel in mouse plasma than those carrying IX-7. In contrast, thespecific clotting activity of IX-6 (495±109%) is 5 times higher thanthat of IX-7 (94±34%). We also evaluate the efficacy of IX-6 whendelivered at lower doses of viral vectors (at 4×10¹¹ vg/kg and 8×10¹⁰vg/kg, respectively). As shown in FIG. 3 b, the specific activitymeasured from mice injected with rAAV2/8 carrying IX-6 reach 1236±418%(4×10¹¹ vg/kg) and 1129±479% (8×10 vg/kg), and the specific activitymeasured in those mice injected with rAAV2/8 carrying IX-7 isapproximately 192±24% (4×10 vg/kg) and 246±116% (8×10 vg/kg) for the twodoses. At the lowest dose, one of the mice injected with IX-6 had 38%normal clotting activity and all 4 mice are above or nearly above the10% therapeutic level. In contrast, the mice injected with IX-7 had only5.6% and 7.4% normal clotting activity. The result further demonstratedthat IX-6 can be an effective reagent when low dose of viral vector ispreferred to reduce the formation of anti-viral antibodies.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A recombinant human Factor IX (FIX) protein comprising the amino acidsequence of SEQ ID NO: 7, except for amino acid substitution(s) at oneor more positions of SEQ ID NO: 7 selected from the group consisting of:substitution at position 86, substitution at position 277, substitutionat positions 86 and 277, substitution at positions 86 and 338,substitution at positions 277 and 338, and substitution at positions 86,277 and
 338. 2. The recombinant human FIX protein of claim 1, whereinthe amino acid substitution(s) is at least one residue selected from thegroup consisting of: alanine, asparagine, aspartic acid, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, methionine,phenylalanine, serine, threonine, tryptophan, tyrosine and valine. 3.The recombinant human FIX protein of claim 2, wherein the residue isalanine.
 4. The recombinant human FIX protein of claim 1, comprising theamino acid sequence of SEQ ID NO:
 6. 5. The recombinant human FIXprotein of claim 1, wherein said protein comprises a 2 fold to 14 foldmore blood clotting activity compared to the wild type human Factor IXprotein.
 6. The recombinant human FIX protein of claim 1, said proteincomprising enhanced affinity with Factor VIIIa.
 7. The recombinant humanFIX protein of claim 4, said protein comprising a K_(d) value of 0.1 to0.5 nM.
 8. The recombinant human FIX protein of claim 4, said proteincomprising a K_(m) value of 32 to 128 nM.
 9. A pharmaceuticalcomposition comprising the recombinant human FIX protein of claim 1 anda pharmaceutically acceptable carrier, excipient or diluent.
 10. Amethod for treating hemophilia comprising administering to a patient inneed thereof an effective amount of the recombinant human FIX protein ofclaim
 1. 11. The method of claim 10, wherein the administering is byinjection.